Latitudinal diversity of biting midge species within the Obsoletus group across three habitats in Europe.

Culicoides species from the Obsoletus group are important vectors of bluetongue and Schmallenberg virus. This group consists of several species that cannot easily be identified using morphological characteristics. Therefore, limited information is available about their distribution and habitat preferences. In this study, we aimed to elucidate the species composition of the Obsoletus group in three habitat types at climatically different latitudes across Europe. Traps were placed in three habitat types in three countries at different latitudes. After DNA extraction, biting midges were identified using PCR and gel electrophoresis. Extraction of DNA using Chelex proved to be a cost and time efficient method for species identification. A latitudinal effect on the relative abundance of species from the Obsoletus group was found. Species composition was unique for most country-habitat combinations. The majority of biting midges were either C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats. Their wide distribution and their high abundance at livestock farms make these species likely candidates for rapid farm-to-farm transmission of pathogens throughout Europe. Our results emphasize the need to differentiate Obsoletus group species to better understand their ecology and contribution to pathogen transmission.

Nucleoside transport across the plasma membrane mediated by equilibrative nucleoside transporter 3 influences metabolism of Arabidopsis seedlings.

The metabolism of nitrogen-rich nucleosides in Arabidopsis seedlings was investigated at the level of import and subsequent salvage or degradation. Uptake and fate of nucleosides imported by equilibrative nucleoside transporter 3 (ENT3) was analysed and, furthermore, a comprehensive analysis of the effect of exogenously fed nucleosides at the level of metabolic as well as transcriptomic alterations was performed. Expression of nucleoside transporters ENT1 and ENT3, together with nucleoside import, was increased upon nitrogen limitation. Thereby a role for ENT3, which is expressed mainly in the vasculature of roots and leaves, as a major import route for nucleosides was supported. Exogenously fed nucleosides were able to attenuate nitrogen starvation effects such as chlorophyll breakdown, anthocyanin accumulation, RNA breakdown and reduced levels of amino acids. In response to nucleoside supply, up-regulation of genes involved in nitrogen distribution in plants was observed. In addition, genes involved in nucleoside metabolism were identified as regulated upon nitrogen limitation. In summary, an overall beneficial effect of nucleoside supply to Arabidopsis seedlings, especially under limiting nitrogen conditions, was observed.

Nucleoside transport and associated metabolism.

Nucleosides are intermediates of nucleotide metabolism. Nucleotide de novo synthesis generates the nucleoside monophosphates AMP and UMP, which are further processed to all purine and pyrimidine nucleotides involved in multiple cellular reactions, including the synthesis of nucleic acids. Catabolism of these substances results in the formation of nucleosides, which are further degraded by nucleoside hydrolase to nucleobases. Both nucleosides and nucleobases can be exchanged between cells and tissues through multiple isoforms of corresponding transport proteins. After uptake into a cell, nucleosides and nucleobases can undergo salvage reactions or catabolism. Whereas energy is preserved by salvage pathway reactions, catabolism liberates ammonia, which is then incorporated into amino acids. Keeping the balance between nitrogen consumption during nucleotide de novo synthesis and ammonia liberation by nucleotide catabolism is essential for correct plant development. Senescence and seed germination represent situations in plant development where marked fluctuations in nucleotide pools occur. Furthermore, extracellular nucleotide metabolism has become an immensely interesting research topic. In addition, selected aspects of nucleoside transport in yeast, protists and humans are discussed.

Characterisation of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At).

Here we report on the isolation of an Arabidopsis thaliana cDNA that is able to complement a Saccharomyces cerevisiae mutant unable to synthesise adenine. This cDNA encodes a highly hydrophobic protein (ENT1,At) of 428 amino acids, showing high similarity to the human nucleoside transporter hENT1. Yeast cells expressing ENT1,At are able to grow on adenosine-containing media, adenosine import exhibited an apparent affinity (K(M)) of 3.6 microM, and led to accumulation of this nucleoside within the yeast cell. Transport is inhibited by various nucleosides. Typical inhibitors of ENT-type nucleoside transporters do not inhibit (3)H-adenosine import. The presence of protonophores abolished adenosine import, indicating that ENT1,At catalyse a proton-dependent adenosine transport. This is the first functional characterisation of a plant nucleoside transport protein.

Two nucleotide transport proteins in Chlamydia trachomatis, one for net nucleoside triphosphate uptake and the other for transport of energy.

The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1Ct and Npt2Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His10-Npt1Ct catalyzed ATP and ADP transport in an exchange mode. The apparent Km values were 48 (ATP) and 39 (ADP) microM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His10-Npt2Ct transported all four ribonucleoside triphosphates with apparent Km values of 31 microM (GTP), 302 microM (UTP), 528 microM (CTP), and 1,158 microM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2Ct. This observation indicated that His10-Npt2Ct acts as a nucleosidetriphosphate/H+ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1Ct provides chlamydiae with energy whereas Npt2Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.

Expression of a plastidic ATP/ADP transporter gene in Escherichia coli leads to a functional adenine nucleotide transport system in the bacterial cytoplasmic membrane.

Recently, a second type of eucaryotic adenine nucleotide transporter located in the inner envelope membrane of higher plants has been identified at the molecular level (Neuhaus, H. E., Thom, E., Möhlmann, T., Steup, M., and Kampfenkel, K. (1997) Plant J. 11, 73-82). Here we have analyzed the biochemical properties of this ATP/ADP transporter from Arabidopsis thaliana (AATP1, At). This analysis was carried out by expressing a cDNA encoding this carrier as a histidine-tagged chimeric protein heterologously in Escherichia coli. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-induced E. coli cells were able to import radioactively labeled [alpha-32P]ATP. Uninduced E. coli cells did not import [alpha-32P]ATP. Further control experiments revealed that IPTG induction did not promote import of other phosphorylated or unphosphorylated metabolites into the bacterial cell indicating the specificity of [alpha-32P]ATP transport. [alpha-32P]ATP uptake into induced E. coli cells was linear with time for several minutes allowing for determination of kinetic constants. The apparent Km for ATP was 17 microM which is close to values reported on the authentic protein in isolated plastids. ADP was a strong competitive inhibitor of -alpha-32P-ATP uptake (Ki ADP 3.6 microM). Other metabolites like AMP, ADP glucose, UTP, UDP, NAD, and NADP did not influence [alpha-32P]ATP uptake. IPTG-induced E. coli cells preloaded with [alpha-32P]ATP exported radioactively labeled adenylates after exogenous addition of unlabeled ATP or ADP indicating a counter exchange mechanism of transport. The biochemical properties of the heterologously expressed AATP1 gene product demonstrated that the protein is functionally integrated in the cytoplasmic membrane of E. coli. This is the first report of the functional expression of a plant membrane protein in E. coli leading to new transport properties across the cytoplasmic membrane. The functional integration of a plant membrane protein in the cytoplasmic membrane of E. coli offers new possibilities for future studies of the structural and mechanistic properties of this transporter. Since IPTG induction allowed synthesis of a 67-kDa protein in E. coli, which was subsequently specifically enriched by metal-chelate chromatography, this procaryotic heterologous expression system might provide a suitable system for overproduction of membrane proteins of eucaryotic origin in the near future.

Occurrence of two plastidic ATP/ADP transporters in Arabidopsis thaliana L.--molecular characterisation and comparative structural analysis of similar ATP/ADP translocators from plastids and Rickettsia prowazekii.

Recently, we sequenced a cDNA clone from Arabidopsis thaliana L. encoding an ATP/ADP transporter protein (AATP1) located in the plastid envelope membrane. The deduced amino acid sequence of AATP1 exhibits a high degree of similarity (> 66%) to the ATP/ADP transporter from the obligate intracellular gram-negative bacterium Rickettsia prowazekii. Here we report a second plastidic ATP/ADP carrier from A. thaliana (AATP2). As deduced from the amino acid sequence, AATP2 exhibits 77.6% identity to AATP1 and 36% to the rickettsial protein. Hydropathy analysis indicates that all three translocators are highly hydrophobic membrane proteins, which exhibit marked similarities and differences. The AATP1 translocator lacks the sixth transmembrane domain that is present in AATP2 and the bacterial transporter in R. prowazekii. In contrast to AATP1 and the bacterial transport protein, only AATP2 exhibits a truncated C-terminal end. To compare the general biochemical properties of AATP2 with the known transport properties of AATP1 we cloned the entire AATP2 cDNA into plasmid pJT118, leading to the presence of an additional N-terminal histidine tag of 10 amino acids. For heterologous expression of His10-AATP2 we chose the Escherichia coli strain C43, which was reported recently to allow overproduction of eucaryotic membrane transport proteins. After transformation and subsequent induction by isopropylthio-2-D-galactopyranoside intact E. coli cells harbouring plasmid pJT118 showed import of radioactively labelled ATP and ADP. As deduced from a Lineweaver-Burk analysis His10-AATP2 exhibited apparent Km values for ATP and ADP of 22 microM and 20 microM, respectively. Import of ADP into His10-AATP2-expressing E. coli cells occurred at a rate of 24 nmol x mg protein(-1) x h(-1), which was about threefold faster than import of ATP. These biochemical characteristics are similar to transport properties of the heterologously expressed His10-AATP1 protein.

ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope.

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395-401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.

Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro, is targeted to envelope membranes of isolated spinach chloroplasts. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations. The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli. In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E. coli. To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter-exchange mode of transport. Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator. An apparent KM for ATP of 28 microM was determined which is similar to values reported for isolated plastids. The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants.

We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. thaliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

Analysis of carbohydrate transport across the envelope of isolated cauliflower-bud amyloplasts.

Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4'-di-isothiocyano-2,2'- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.